Method and device for multiplying and differentiating cells in the presence of growth factors and of a biological matrix or of a supporting structure

ABSTRACT

The invention relates to an in-vitro and in-vivo method for multiplying and differentiating cells, during which the growth process of the cells is initiated or terminated and structurally directed by the use of growth factors thrombopoietin (TPO) and/or erythropoietin (EPO), and/or growth hormone (GH), particularly human growth hormone (HGH), and/or somatostatin and/or leukemia inhibitor factor (LHF) and/or ciliary neurotropic factor (CNTF). The invention relates to a biological matrix or supporting structure containing the aforementioned growth factors, and to a method and device for the production thereof and for carrying out the inventive method.

The present invention relates to the use of at least one growth factorin isolated form for the cultivation of primarily differentiated cells,for the locally specific and/or directed differentiation of adult cellsand/or for the regeneration of bones, tissues and/or endocrine organs.

In ontogenesis, that is to say the development of the individualorganism, there is expression of growth factors which are able toinitiate fundamental structural processes which are numerical inrelation to the number of cells. In the growing organism, the abilityfor structural repairs through regeneration is increasingly lost becausethese growth factors are no longer expressed. Factors of the bone marrowand of the blood-forming organs are coupled in time to growth processesof other organs during specific ontogenetic processes.

One disadvantage of known growth factors such as, for example, epidermalgrowth factor (EGF), vascular endothelial growth factor (VEGF) orhepatocyte growth factor (HGF) is that the multiplication processes,especially on use of primary cells in vitro, are limited and that theuse in vivo is problematic because of possible side effects such as, forexample, the activation of oncogenes.

It has to date been assumed that tissue extracts such as, for example,from the pituitary or the hypothalamus are particularly suitable forbringing about multiplication of hepatocyte cells (see, for example,U.S. Pat. No. 6,008,047). Such animal or, occasionally, human extractshave already been added to cell cultures. The use of animal or humantissue extracts is, however, problematic in laboratory work or inclinical use owing to transmissible viral diseases such as, for example,BSE, pig or sheep viruses. The use of such extracts demonstrates thelack of knowledge about the actually relevant factors and theirpotential uses and effects. A further substantial disadvantage is thatthrough such heterogeneous extracts, which are generally difficult todefine and depend considerably on the source used, there is alsointroduction into the culture of factors which, in some circumstances,bring about unwanted side effects or properties on clinical use.Accurate knowledge of the factors and controlled dosage thereof wouldtherefore be both an important factor for being able to multiply anddifferentiate cells, especially in the area of tissue engineering,appropriately, and for inducing structural processes ofthree-dimensional (3-D) regeneration.

Such structural tasks are a priority in particular for tissueengineering, although 3-D growth and its initiation is not as yetunderstood. Although conventional approaches such as aggregate culturesachieve a high density, they must be built up with cells which have beenpreexpanded or isolated from primary tissues, e.g. hepatocytes. Aninductive growth process into a defined, predetermined structure has notto date been possible.

On the contrary, cells such as, for example, hepatocytes are stillembedded after the multiplication phases in a gel in order to avoid theformation of further, also large, aggregates (superaggregates). Thesegels are two-dimensional in extent, comprise a high cell density andtherefore stop cell multiplication. Such 2-D gel inclusions, whichresult in layers, have already been described by Bader et al. (1995),Artif. Organs, 19, 368-374, as sandwich model or gel entrapment.Although the embedding of aggregates in gels results in an improvementin maintenance of differentiation, it does not result in further growth.

A shape-creating growth from a few precursor cells to a 3-D structureand an inductive behavior for neighborhood processes in the sense oftissue regeneration in vitro and in vivo has not to date been possible.However, inductive growth behavior of cells means a considerableinnovation in particular for therapeutic or biotechnological processes.Such a growth behavior should, assisted by a 3-D supporting matrix,allow growth not only in the sense of colonization or structuralremodeling but in fact be able to allow directed de novo formation froman induction nucleus. Such processes take place in ontogenesis and buildupon a pre-existing anlage.

It is known merely that growth factors, especially in the case ofneuronal progenitors of fetal origin such as, for example, leukemiainhibitory factor (LIF), ciliary neurotropic factor (CNTF), glialderived neurotrophic factor (GDNF) or nerve growth factor (NGF), make aproliferation phase of undifferentiated neurons possible. However, afterdifferentiation is achieved, these factors are no longer able to act.

In tissue engineering there is in addition the problem thatpatient-specific adult cell systems which are already differentiatedfurther than fetal cells are used. In addition, coculture situationsapply in situ and in vitro but are not taken into account inconventional usage. On the contrary, attempts are even made for exampleto avoid cocultures of endothelial cells, macrophages and fibroblasts,as occur in the liver, on expansion of the parenchymal liver cells,because they are unwanted. However, it is now known that the presence ofthese so-called non-parenchymal cells in differentiated cultures make asubstantial contribution to the differentiation.

It is therefore desirable to provide a multiplication method in vitroand/or a regeneration method in vivo which is able substantially tomaintain the physiological state of the cell systems and makesubstantially structural growth possible.

It has now been found that the use of the growth factors thrombopoietin(TPO) and/or erythropoietin (EPO) and/or growth hormone (GH), and/orsomatostatin and/or leukemia inhibitory factor (LIF) and/or ciliaryneurotropic factor (CNTF) initiates and terminates, and structurallyguides, the multiplication and differentiation of cells.

Surprisingly, this has brought about not only a multiplication of cellsbut also an induction of structural processes, in particular a locallyspecific cell multiplication and directed differentiation is broughtabout by an inductive effect on an implant in place (in situ) forexample via a so-called homing process. This means that the growthhormones are able to induce but also terminate these structuralprocesses.

The invention therefore relates to a method for multiplying anddifferentiating cells in vitro, in which the growth process of the cellsis initiated and terminated, and structurally guided, by the use of thegrowth factors TPO and/or EPO and/or GH, especially HGH and/orsomatostatin and/or LIF and/or CNTF.

Thus, TPO is also known for example as c-Mpl ligand, mpl ligand,megapoietin or megakariocyte growth and development factor and has todate not been employed in the culturing of, for example, adulthepatocytes or other primary cells apart from platelets and theirprecursors. TPO is essentially necessary for the development andproliferation of megakariocytes and platelets and thus for the formationof blood platelets. TPO is constitutively produced in the liver and inthe kidneys as 332 amino acid-long protein.

Additional growth factors which can be employed according to the presentinvention are transforming growth factor beta (TGF beta),prostaglandins, granulocyte-macrophage stimulating factor (GM-CSF),growth hormone releasing hormone (GHRH), thyrotropin-releasing hormone(TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasinghormone (CRH), dopamine, antidiuretic hormone (ADH), oxytocin,prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and/orvasopressin.

Besides cessation or reduction of the supply of the described growthfactors to the culture, somatostatin and/or TGF beta and/orprostaglandins are also suitable for terminating the growth process ofthe invention.

The individual concentrations of the growth factors in solution arenormally about 1 to about 100 ng/ml, preferably about 10 to about 50ng/ml, in particular about 10 to about 20 ng/ml. However, in the case oflocal coatings, the concentrations of the growth factors may also be amultiple thereof.

For example, in the case of regeneration of endocrine organs,interaction of the growth factors, in particular of growth hormonereleasing hormone (GHRH), thyrotropin-releasing hormone (TRH),gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone(CRH), somatostatin, dopamines, antidiuretic hormone (ADH) and/oroxytocin, with the supporting matrix may induce endocrinedifferentiation and/or growth in situ.

It is additionally possible to employ prolactin, adrenocorticotropin,beta-celltropin, lutrotropin and/or vasopressin for the structuralprocesses.

In a further embodiment it is additionally possible to employ one ormore nerve regeneration factors, preferably nerve growth factor (NGF)and/or one or more vessel regeneration factors, preferably vascularendothelial growth factor (VEGF) and/or platelet derived growth factor(PDGF).

In the presence of endothelial cells it is possible to achieve anendothelialization of the cells and thus an optimal hemocompatibility.

Said growth factors can generally be purchased commercially but can alsobe prepared by gene manipulation by methods known to the skilled worker.They include not only the naturally occurring growth factors but alsoderivatives or variants having substantially the same biologicalactivity.

Thus, for example, TPO can be purchased commercially from CellSystemsGmbH, St Katharinen. The use of human TPO is preferred for cultivatinghuman adult hepatocytes. In addition, the preparation andcharacterization of TPO and its variants is described for example in EP1 201 246, WO 95/21919, WO 95/21920 and WO 95/26746.

Suitable TPO variants are the TPO derivatives described in WO 95/21919or the allelic variants or species homologs described in WO 95/21920 orthe pegylated TPO described in WO 95/26746 and EP 1 201 246, withoutrestriction thereto. Pegylated TPO means for the purposes of the presentinvention TPO derivatives which are linked to an organic polymer suchas, for example, polyethylene glycol, polypropylene glycol orpolyoxyalkylene. Further variants of TPO also mean derivatives of TPOwhich have a sequence identity of less than 100% and nevertheless havethe activity of TPO, as described preferably in EP 1 201 246. TPOderivatives normally have a sequence identity of at least 70%,preferably at least 75%, especially at least 80% and in particular atleast 85% compared with human TPO including fragments thereof having TPOactivity. A particularly preferred TPO activity for the purposes of thepresent invention is the speeding up of proliferation, differentiationand/or maturation of megakaryocytes or megakaryocyte precursors inplatelet-producing forms of these cells by TPO or its variants.

EPO is also referred to as embryonic form of TPO and is described withits variants for example in EP 0 148 605, EP 0 205 564, EP 0 209 539, EP0 267 678 or EP 0 411 678.

The examples of derivatives and variants described in detail above applyanalogously also to the other growth factors mentioned.

The term growth factor is accordingly not restricted according to thepresent invention to the naturally occurring forms, but also includesnon-naturally occurring forms and variants or derivatives. The termgrowth factor includes according to the present invention not onlygrowth promoters but also growth inhibitors such as, for example,somatostatin, TGF beta and/or prostaglandins. Such growth inhibitors areparticularly suitable for suppressing or inhibiting the growth ofmutated cells such as, for example, tumor cells, by highly concentratedlocal use thereof simultaneously or sequentially, for example also bymeans of hydrogels or slow-release materials.

The growth process of the invention is carried out in a culture suitablefor the particular cells. It is possible in this connection by means ofa suitable device for the cell aggregates formed where appropriateduring the growth process to be broken up and, where appropriate,encapsulated and, where appropriate, frozen.

An example of a suitable device is a grid having, for example, a cuttingmesh structure for example 500 μm in size, which has the effect that newsubsidiary aggregates of, for example, hepatocytes can be repeatedlyproduced. This can advantageously take place in a completely closedsystem. It is possible in particular to employ contactless,automatically or manually controlled pumping systems which consist forexample of piston pumps or generate directed flows generatedmagnetically or by compressed air compression of tubings. In thepresence of endothelial cells it is possible through the shear stress ina perfused bioreactor for spontaneous confluence of the endothelialcells on the surfaces of the aggregates to occur, which may beadvantageous for further use.

Materials suitable for the encapsulation are suitable ones which areknown to the skilled worker and in which, for example, structured shapesor spaces are integrated and make an in situ growth structure orenlargement possible. An alternative possibility is for the capsule tobe dispensed with and, for example, an endothelialization and thusoptimal hemocompatibility to be achieved in the presence of endothelialcells.

In a further embodiment, the growth process of the cells is locallyinitiated and terminated, and structurally guided, preferably by abiological matrix.

The biological matrix is in this case for example treated with one ofsaid growth factors or with a combination of said growth factors asmixture or sequentially. This makes 3-D regeneration and/or artificialguidance of tissue repair or tissue culturing possible even with adultcell systems.

The biological matrix is normally an implant, e.g. a stent, a patch or acatheter, a transplant, e.g. a skin transplant and/or a supportingmaterial for the growth of cells, e.g. a so-called slow releasematerial, e.g. a hydrogel for example based on fibrin and/or polymerssuch as, for example, polylactide or polyhydroxy-alkanoate, and/oralginates, a bone substitute material, e.g. tricalcium phosphate, anallogeneic, autologous or xeinogeneic acellularized or non-acellularizedtissue, e.g. a heart valve, venous valve, arterial valve, skin, vessel,aorta, tendon, comea, cartilage, bones, tracea, nerve, miniscus,intervertebral disc, ureters, urethra or bladder (see, for example, EP 0989 867 or EP 1 172 120), a matrix such as, for example, a laminin,collagen IV and/or Matrigel matrix, preferably a feeder layer such as,for example, collagen I, 3T3 and/or MRC-5 feeder layer, or a collagenfabric.

In a further preferred embodiment, the biological matrix is precolonizedwith cells, preferably tissue-specific cells, precursor cells, bonemarrow cells, peripheral blood, adipose tissue and/or fibrous tissue,e.g. with adult precursor cells from the bone marrow, by methods knownto the skilled worker. It is possible in this way to achieveanticipation of the in vivo wound-healing process in vitro, and thus ashortened reintegration time takes place after implantation in vivo.

The cells used according to the present invention are in particularadult cells, i.e. primarily differentiated cells which preferably nolonger have an embryonic or fetal phenotype, particularly preferablyhuman adult cells. Examples thereof axe adult progenitor cells,tissue-specific cells, preferably osteoblasts, fibroblasts, hepatocytesand/or smooth muscle cells.

However, it is also possible to suppress or inhibit mutated cells suchas, for example, tumor cells, by for example highly concentrated,simultaneous or sequential dosage of growth inhibitors such assomatostatin, TGF beta and/or prostaglandins. It is possible in thiscase to employ the hydrogels or slow-release materials which havealready been mentioned and which comprise at least one of said growthinhibitors or are supplemented therewith, and are applied locally or inthe vicinity of the mutated cells.

The method of the invention is thus particularly suitable for locallyspecific and/or directed multiplication, structural growth andsubsequent differentiation of adult cells and/or for the regeneration ofbones, tissues and/or endocrine organs, e.g. of heart valves, venousvalves, arterial valves, skin, vessels, aortas, tendons, comea,cartilage, bones, tracea, nerves, miniscus, intervertebral disc,ureters, urethra or bladders.

The method of the invention can also be employed for localadministration in vivo by said growth factors being employed eitheralone or in combination as mixture or sequentially, or in combinationwith said biological matrices or supporting structures, for example fortissue regeneration, such as, for example, liver regeneration,myocardical regeneration or for wound healing in the region of the skin,e.g. for diabetic ulcers, or gingiva. For example, it is possible for,for example, TPO to be applied in a hydrogel, e.g. fibrin and/or apolymer such as, for example, polylactide or polyhydroxyalkanoate,and/or an alginate, to the resection surface for example of a liver forliver regeneration, or to be administered locally or systemically in,for example, acute liver failure via a port with the aid of a catheter.Said growth factors can thus be administered for example before, duringor after a liver resection or removal of liver tissue in order to assistliver regeneration. On use of said growth factors for promotingcartilage regeneration, the growth factor(s) can be injected directlyinto the knee joint. It is thus possible for the growth factor(s) to actvia the sinovial fluid directly on the formation of a new cartilagestructure.

Consequently, the present invention also relates to the use of thegrowth factors TPO and/or EPO and/or GH and/or somatostatin and/or LIFand/or CNTF for producing a medicament for the treatment of regenerationof bones, cartilage, tissues and/or endocrine organs, e.g. parenchymaland/or non-parenchymal organs, especially of myocardium, heart valves,venous valves, arterial valves, skin, vessels, aortas, tendons, comea,cartilage, bones, tracea, nerves, miniscus, intervertebral disc, liver,intestinal epithelium, ureters, urethra or bladders, or for thetreatment of degenerative disorders and/or for assisting the woundhealing process, especially in Crohn's disease, ulcerative colitisand/or in the region of the skin, preferably for diabetic ulcers orgingiva and/or for the treatment of liver disorders, especially ofcirrhosis of the liver, hepatitis, acute or chronic liver failure and/orwound healing in the muscle region after sports injuries, muscledisorders, bone injuries, soft tissue injuries and/or for improvingwound healing and tissue regeneration, e.g. after operations, acute andchronic disorders and/or for improving wound healing and tissueregeneration, for example after operations, acute and chronic disordersand/or ischemic myocardial disorders for stimulating neoangiogenesis andregeneration and/or ischemias after injuries and trauma and/orregeneration of tissues following a tissue injury, e.g. with myocardialinfarction or thromboses (central or peripheral) in some circumstanceswith subsequent ischemia. EPO dosage in this case makes neoangiogenesisand subsequent or accompanying tissue regeneration possible.

In a particular embodiment there is use as growth factor in addition oftransforming growth factor beta (TGF beta), prostaglandins,granulocyte-macrophage stimulating factor (GM-CSF), growth hormonereleasing hormone (GHRH), thyrotropin-releasing hormone (TRH),gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone(CRH), dopamine, antidiuretic hormone (ADH), oxytocin, prolactin,adrenocorticotropin, beta-celltropin, lutrotropin and/or vasopressin, oradditionally of one or more nerve regeneration factors, preferably nervegrowth factor (NGF) and/or one or more vessel regeneration factors,preferably vascular endothelial growth factor (VEGF) and/or plateletderived growth factor (PDGF).

The further embodiments described in the present invention applyanalogously also to the described uses of the invention.

A further possibility is for a biological matrix or supporting structurecomprising at least one of the growth factors TPO, EPO, GH, especiallyHGH, somatostatin, LIF and/or CNTF, to be used as inductive substratefor 3-D growth and/or regeneration within a multiplication phase orafter a multiplication phase for differentiation or for growth arrest.For example, at least one of said growth factors can be applied to astent in combination with a so-called slow-release material, asdescribed by way of example above.

The present invention therefore relates further also to a biologicalmatrix or supporting structure comprising at least one of the growthfactors thrombopoietin (TPO), erythropoietin (EPO), growth factor (GH),especially human growth hormone (HGH), somatostatin, leukemia inhibitoryfactor (LIF) and/or ciliary neurotropic factor (CNTF), where thebiological matrix or supporting structure in this may also additionallycomprise at least one of the growth factors TGF beta, prostaglandin,GM-CSF, GHRH, TRH, GnRH, CRH, dopamine, ADH, oxytocin, prolactin,adrenocorticotropin, beta-celltropin, lutrotropin and/or vasopressinand, where appropriate, additionally one or more nerve regenerationfactors, preferably nerve growth factor (NGF) and/or one or more vesselregeneration factors, preferably vascular endothelial growth factor(VEGF) and/or platelet derived growth factor (PDGF).

The biological matrix or supporting structure of the invention is, forexample, an implant, a transplant and/or a supporting material for thegrowth of cells, the biological matrix or supporting structure possiblybeing a stent, a catheter, a skin, a hydrogel, a bone substitutematerial, an allogeneic, autologous or xenogeneic, acellularized ornon-acellularized tissue, a synthetic tissue, a feeder layer or a fabricsuch as, for example, a fabric made of collagen, laminin and/orfibronectin with or without synthetic or other type of basic structure,such as, for example, plastic or a biological matrix. Exemplaryembodiments have already been described above.

The biological matrix or supporting structure is, as already describedabove in detail, preferably already precolonized with tissue-specificcells, precursor cells, bone marrow cells, peripheral blood, adiposetissue and/or fibrous tissue, or already prepared for in vivocolonization or inductive remodeling in vitro.

The biological matrix or supporting structure can also be coated with a(bio)polymer layer which comprises at least one of said growth factors.Fibrin, plasma, collagen and/or polylactides are suitable for example as(bio)polymer layer.

The present invention also relates to a method for producing abiological matrix or supporting structure of the invention, in which anoptionally activated biological matrix or supporting structure is coatedwith at least one of the growth factors TPO, EPO, GH, in particular HGH,somatostatin, LIF and/or CNTF, where said matrix or supporting structurecan optionally be coated with additionally at least one of the growthfactors TGF beta, prostaglandin, GM-CSF, GHRH, TRH, GnRH, CRH, dopamine,ADH, oxytocin, prolactin, adrenocorticotropin, beta-celltropin,lutrotropin and/or vasopressin and, where appropriate, additionally withone or more nerve regeneration factors, preferably NGF and/or one ormore vessel regeneration factors, preferably VEGF and/or PDGF.

The activation of the biological matrix or supporting structure can takeplace for example by means of plasma ionization, e.g. using hydrogenperoxide, or by means of laser activation.

An alternative possibility is a coating with a biodegradable(bio)polymer layer which comprises said growth factor(s). Suitableexamples for this purpose are fibrin, plasma, blood, collagen and/orpolylactides.

It is likewise possible in the method of the invention for thebiological matrix or supporting structure to be precolonized in vitrowith cells, preferably tissue-specific cells, precursor cells, bonemarrow cells, peripheral blood, adipose tissue and/or fibrous tissue.

The preferred features or feature examples of the present inventionwhich are described above apply analogously to the production process ofthe invention.

The present invention also extends to a device for carrying out themethod of the invention, where a perfused bioreactor, especially in theform of a closed system, is preferred.

The following examples are intended to explain the invention in detailwithout restricting it.

EXAMPLES

1. Bone Regeneration

A single-phase beta tricalcium phosphate is prepared as granules with amicroporosity of, for example, >15 μm and shaped in a mold of a 3-Ddefect corresponding to a patient's requirement. This normally takesplace in a sintering process. The material is subsequently treated byplasma ionization so that activation of the surfaces occurs and theconstruct is placed in a solution with thrombopoietin, erythropoietinand/or growth hormone (GH) and thus coated in small quantities in adefined way. Alternatively, an incubation in a solution without previoussurface activation or a coating with a biodegradable (bio)polymer layercomprising these growth factors can take place. It is possible in thiscase to employ for example fibrin, plasma, collagen and/or polylactides.

This construct is then either immediately introduced into a defect orprecolonized in vitro with tissue-specific cells, precursor cells orbone marrow cells. This achieves anticipation of the in vivowound-healing process in vitro and thus a shortened reintegration timecan take place after implantation in vivo (e.g. after 7 days). Acombination with factors of nerve regeneration (NOF) or vesselregeneration (VEGF, PDGF) is possible. Combination with thestructure-forming factors and environment concepts is of interest inthis connection.

In vivo and in vitro there is integration of the blood-forming and stemcell-rich bone marrow and an increased rate of differentiation ofosteoblasts and an increased rate of absorption of the supporting matrixand a replacement by normal bone. Site-specific integration takes placeowing to the recruitment competence and the inductive character.

This can be further promoted by colonization on the external sides withperiosteum in vitro.

2. Heart Valve Regeneration and Production of Urological Constructs

A biological matrix (allogeneic or autologous heart valve with andwithout acellularization, a synthetic supporting structure made ofplastics which resembles the physiological microenvironment of thecardiovascular target tissue in terms of the chemical composition of thecollagens and their spatial arrangement) is precoated withthrombopoietin and erythropoietin as growth factors.

The material is then treated by plasma ionization (e.g. using hydrogenperoxide, H₂O₂), simultaneously achieving sterilization, so thatactivation of the surfaces occurs and the construct is placed in asolution with thrombopoietin, erythropoietin and/or growth hormone (GH)and thus coated in small quantities in a defined way. Alternatively,incubation in a solution without previous surface activation or coatingwith a biodegradable (bio)polymer layer comprising these growth factorsis possible. Fibrin, plasma, blood, collagen or polylactides can beemployed in this case.

This construct is then either immediately introduced at the requiredsite (heart valve position, as patch or vessel replacement) orprecolonized in vitro with tissue-specific cells, precursor cells orbone marrow cells. This achieves anticipation of the in vivowound-healing process in vitro and thus a shortened reintegration timecan take place after implantation in vivo (e.g. after 7 days). Acombination with factors of nerve regeneration (NGF) or vesselregeneration (VEGF, PDGF) is possible, but not absolutely necessary.

In vivo and in vitro there is integration of the blood-forming and stemcell-rich bone marrow and an increased rate of differentiation offibroblasts and smooth muscle cells and an increased rate of absorptionof the supporting matrix and a replacement by normal cardiovasculartissue. Site-specific integration takes place owing to the recruitmentcompetence and the inductive character.

This can be further promoted by colonization on the external sides withendothelial cells in vitro.

Urological constructs can be produced in a corresponding manner.

3. Multiplication of Adult Hepatocytes in Coculture with NonparenohymalCells

A mixed liver cell population from a biopsy or a partial sectate aretreated with TPO and/or EPO and/or growth hormone, e.g. HGH in aconcentration of 10-50 ng/ml by addition to the medium supernatant. Theseeding cell density is 10 000 cells/cm. After confluence is reached,the cells are treated with 0.005% collagenase and 0.01% trypsin with theaddition of 2 g/l albumin or autologous serum (10-20%) for 5 h. Thecells are then aspirated off and washed three times in culture medium(Williams B (Williams et al. (1971) Exptl. Cell Res., 69, 106) with 2g/l albumin and then put for sedimentation in a collagen-coated Petridish.

Differentiation of the cells can be achieved by overlayering with anextracellular matrix.

Alternatively, the cells can be prevented from sedimenting by agitationand come together for the aggregation.

In order to avoid too great an enlargement of the aggregates during thegrowth process, the cells can be paused in an appropriate device over agrid having a cutting mesh structure 500 μm in size, so that newsubsidiary aggregates can be repeatedly produced. This can take place ina completely closed system. Ideally, contactless pumping systems (nosqueezing by peristaltic systems but directed flows generatedmagnetically or by compressed air compression of tubings, or pistonpumps—automatic or manual) are employed.

The cells can then be encapsulated and frozen. Structured shapes andspaces can be integrated in the capsule structure, which makes an insitu growth structure and enlargement possible.

Alternatively, the capsule can be dispensed with and, through thepresence of the endothelial cells in this system and targeted additionof these cells, an endothelialization and thus optimal hemocompatibilitycan be achieved.

The shear stress in a perfused bioreactor results in spontaneousconfluence of the endothelial cells on the surfaces of the aggregates.When the target size is reached, they can be frozen for example in thebags which are already ideally used for the culture.

4. Soft Tissues (Muscle Patches, Nerves, Tendons)

For reconstructing abdominal wall defects it is possible to producecollagen tile or fabrics such as laminin, fibronectin with or withoutsynthetic or another type of basic structure such as, for example,plastic or a biological matrix, or spatially defined structures (tubesfor nerves, tendons) correspondingly as above. These collagen tile orstructures are shaped, coated with TPO, EPO and/or growth hormone (GH)and implanted or precolonized with cells of the target tissue (e.g.tenocytes, neurons).

A biological matrix (allogeneic or autologous heart valve with andwithout acellularization, a synthetic supporting structure made ofplastics which resembles the physiological microenvironment of thetarget tissue in terms of the chemical composition of the collagens andtheir spatial arrangement) is precoated with thrombopoietin anderythropoietin as growth factors.

Subsequent or prior to this the material is then treated by plasmaionization (e.g. using hydrogen peroxide, H₂O₂), simultaneouslyachieving sterilization, so that activation of the surfaces occurs andthe construct is placed in a solution with thrombopoietin,erythropoietin and/or growth hormone and thus coated in small quantitiesin a defined way. Alternatively, incubation in a solution withoutprevious surface activation or coating with a biodegradable (bio)polymerlayer comprising these growth factors is possible. Fibrin, plasma,collagen and/or polylactides can be employed in this case.

This construct is then either immediately introduced at the requiredsite (abdominal wall, myocardium, skeletal muscle as patch) orprecolonized in vitro with tissue-specific cells, precursor cells orbone marrow cells. This achieves anticipation of the in vivowound-healing process in vitro and thus a shortened reintegration timecan take place after implantation in vivo (e.g. after 7 days). Acombination with factors of nerve regeneration (NGF) or vesselregeneration (VEGF, PDGF) is possible, but not absolutely necessary.

In vivo and in vitro there is integration of the blood-forming and stemcell-rich bone marrow and an increased rate of differentiation offibroblasts and smooth muscle cells and an increased rate of absorptionof the supporting matrix and a replacement by normal cardiovasculartissue. Site-specific integration takes place owing to the recruitmentcompetence and the inductive character.

This can be further promoted by in vitro colonization on the externalsides with keratinocysts (abdominal muscle), Schwann's cells and/orfibrous tissue.

5. Regeneration of Tissues In Vivo

a) Liver

After partial resection of the liver, EPO is administered systemicallyand/or topically to the patient by application to the resection surfacein conjunction with a polymer. The polymer may be a biopolymer such as,for example, fibrin (from, for example, fibrin glue), polymerizedplasma, polymerized blood or bioadhesives, e.g. mussel adhesive.However, it may also be synthetic or biological gels or hydrogels. TheEPO can also be introduced into fabrics which serve to stop bleeding(e.g. collagen fabrics, tamponade, wovens and knits).

Through the action of EPO there is restoration of the original volume ofthe liver within 2 weeks. This involves not only a multiplication of thehepatocytes but also a coordinated growth in which the vessels, the bileducts and the capsular structures also grow back to their original size.

It was possible to show in 30 animals that regeneration of the livertook place significantly compared with the control animals (without EPOdosage).

EPO can also be employed for regenerating the liver in chronic liverdisorders such as, for example, cirrhosis, fibrosis, hepatitis. It isthus possible for the first time to achieve a therapeutic effect inrelation to the liver parenchyma.

b) Inflammatory Bowel Disorders

In patients with Crohn's disease, wound healing in the region of theintestinal epithelium is impaired. Underlying tissue structures may alsobe involved in inflammatory reactions. In these patients, systemicand/or topical dosage of EPO leads to restoration of the intestinalepithelium through regeneration. Topical dosage may take place by slowrelease capsules in the intestinal region or by giving suppositorieswith gels or local installation with solutions.

Absorption in the regional vascular area can be optimized by givingpegylated (PEG) compounds, so that systemic effect and thus initiationof the wound-healing process can take place via the regional dosage inthe area of inflammation.

The presence of an anemia is to be regarded as a prognostic positivefactor for patients with Crohn's disease. It was assumed in the pastthat the anemia is an independent concomitant disorder or isattributable to the wasting due to absorption problems. Our results showthat the impairment of wound healing involves a deficiency of endogenousEPO. It is thus possible to treat Crohn's disease very selectively byexogenous dosage of EPO. Further uses are to be found also in the areaof ulcerative colitis.

c) Impairments or Wound Healing in the Region of the Skin

Patients with diabetic ulcers have trophic disorders which make woundclosure in the region usually of the legs difficult. The capacity forstructural tissue regeneration is restricted owing to the basicdisorder. In these cases, wound healing is induced through systemicand/or topical dosage of EPO. It proves to be advantageous to administerEPO after roughening of the lower stratum during a debridement. Thecombination of EPO with a polymerization induced by calcium chlorideleads to integration of EPO in a blood clot, resulting in a topicalslow-release preparation. Alternatively, EPO can also be administered inconjunction with a fibrin glue or with a fabric or with a tamponade(e.g. collagen fabric) impregnated with EPO.

EPO can be given in a similar manner for all other wound healingrequirements, e.g. in the muscle region after sports injuries, muscledisorders, bone injuries, soft-tissue injuries and generally forimproving wound healing and tissue regeneration, e.g. after operations,acute and chronic disorders.

1-27. (canceled)
 28. A method for in vitro regeneration comprising the following steps: provision of a liver sectate in vitro, induction of a significant structural growth of the sectate compared with an untreated sectate(control) through administration of EFO, TPO, GH or derivatives thereof on the liver resection surface; and where appropriate, use of the treated sectate for the treatment of liver disorders.
 29. The method as claimed in claim 28 for multiplying and differentiating cells in vitro, characterized in that the growth process of the cells is initiated and terminated, and structurally guided, through the use of the growth factors thrombopoietin (TPO) and/or erythropoietin (EPO), and/or growth hormone (GH), in particular human growth hormone (HGH), and/or somatostatin and/or leukemia inhibitory factor (LIP) and/or ciliary neurotropic factor (CNTF).
 30. The method as claimed in claim 29, characterized in that transforming growth factor beta (TGF beta), prostaglandin, granulocyte-macrophage stimulating factor (GM-CSF), growth hormone releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH), dopamine, antidiuretic hormone (ADH), oxytocin, prolactin, adrenocorticotropin, beta-celitropin, lutrotropin and/or vasopressin is employed additionally as growth factor.
 31. The method as claimed in claim 29 or 30, characterized in that one or more nerve regeneration factors, preferably nerve growth factor (NGF) and/or one or more vessel regeneration factors, preferably vascular endothelial growth factor (VEGF) and/or platelet derived growth factor (PDGF) are employed in addition.
 32. The method as claimed in at least one of claims 29-31, characterized in that the method is carried out in the presence of endothelial cells.
 33. The method as claimed in at least one of claims 29 to 32, characterized in that the growth process of the cells is locally initiated and terminated, and structurally guided.
 34. The method as claimed in claim 33, characterized in that the growth process of the cells is locally initiated and terminated, and structurally guided, by a biological matrix or by a supporting structure.
 35. The method as claimed in claim 34, characterized in that the biological matrix or supporting structure is treated with one of said growth factors or with a combination of said growth factors as mixture or sequentially.
 36. The method as claimed in claim 34 or 35, characterized in that an implant, a transplant and/or a supporting material is used as biological matrix or as supporting structure for the growth of cells.
 37. The method as claimed in at least one of claims 29 to 36, characterized in that the biological matrix or supporting structure has been precolonized with cells, preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and/or fibrous tissue, or already prepared in vitro for the in vivo colonization or the inductive remodeling.
 38. The method as claimed in at least one of claims 29-37, characterized in that adult progenitor cells and/or tissue-specific cells, preferably osteoblasts, fibroblasts, hepatocytes and/or smooth muscle cells, are employed as cells.
 39. The method as claimed in at least one of claims 29-38 for locally specific and/or directed multiplication, structural growth and subsequent differentiation of adult cells and/or for regeneration of bones, tissues and/or endocrine organs.
 40. The method as claimed in at least one of claims 29 to 32, characterized in that the cell aggregates which form where appropriate during the growth process are broken up and, where appropriate, encapsulated and, where appropriate, frozen by means of a suitable device.
 41. A biological matrix or supporting structure comprising at least one of the growth factors TPO, EPO, GH, especially HGH, somatostatin, LIF and/or CNTF.
 42. The biological matrix or supporting structure as claimed in claim 41, additionally comprising at least one of the growth factors TGF beta, prostaglandins, GM-CSF, GHRH, TRH, GnRH, CRH, dopamine, ADH, oxytocin, prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and/or vasopressin and, where appropriate, additionally one or more nerve regeneration factors, preferably NGF and/or one or more vessel regeneration factors, preferably VEGF and/or PDGF.
 43. The biological matrix or supporting structure as claimed in claim 41 or 42, characterized in that the biological matrix or supporting structure is an implant, a transplant and/or a supporting material for the growth of cells.
 44. The biological matrix or supporting structure as claimed in any of claims 41 to 43, characterized in that the biological matrix or supporting structure is a stent, a patch, a catheter, a skin, a hydrogel, a bone substitute material, an allogeneic, autologous or xenogeneic, acellularized or non-acellularized tissue, a synthetic tissue, a feeder layer or a fabric.
 45. The biological matrix or supporting structure as claimed in any of claims 41 to 44, characterized in that the biological matrix or supporting structure is precolonized with cells, preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and/or fibrous tissue.
 46. The biological matrix or supporting structure as claimed in any of claims 41 to 45, characterized in that the biological matrix or supporting structure is coated with a biodegradable (bio)polymer layer comprising at least one of said growth factors.
 47. A method for producing a biological matrix or supporting structure as claimed in at least one of claims 41 to 46, characterized in that an optionally activated biological matrix or supporting structure is coated with at least one of the growth factors TPO, EPO, GH, especially HGH, somatostatin, LIF and/or CNTF.
 48. The method as claimed in claim 47, characterized in that said matrix or supporting structure is coated with additionally at least one of the growth factors TGF beta, prostaglandin, GM-CSF, GHRH, TRH, GnRH, CRM, dopamine, ADH, oxytocin, prolactin, adrenocorticotropin, beta-celitropin, lutrotropin and/or vasopressin and, where appropriate, additionally with one or more nerve regeneration factors, preferably NGF and/or one or more vessel regeneration factors, preferably VEGF and/or PDGF.
 49. The method as claimed in claim 47 or 48, characterized in that the biological matrix or supporting structure is activated by means of plasma ionization or laser activation.
 50. The method as claimed in at least one of claims 47 to 49, characterized in that said biological matrix or supporting structure is precolonized in vitro with cells, preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and/or fibrous tissue.
 51. A device for carrying out a method as claimed in at least one of claims 29 to 40 and 48 to
 50. 52. The device as claimed in claim 51, characterized in that the device is a perfused bioreactor, preferably in the form of a closed system. 